This invention relates to nucleic acid hybridization methods.
Nucleic acid hybridization using nitrocellulose filters and other planar, solid supports is used to identify specific DNA or RNA sequences. Colony hybridizations in which colonies of organisms are transferred from a culture plate to nitrocellulose filters have been used to identify bacteria, bacteriophages, other organisms, genes within organisms, RNA transcriptional products, recombinant DNA clones, etc. The colony hybridization method from which many frequently used procedures are derived is that of Grunstein and Hogness, 72 Proc. Natl. Acad. Sci. 3961 (1975). A review of the use of nucleic acid filter hybridization procedures in modern molecular biology research is given in Maniatis et al., Molecular Cloning - A Laboratory Manual 312-61 (Cold Spring Harbor Laboratory, New York 1982). A review of nucleic acid filter hybridizations in medical diagnostics is given in Meinkoth et al., 138 Analytic Biochem. 267-84 (1984).
While the standard filter hydridization procedure as practiced by Grunstein and Hogness and many others does provide a sensitive and very specific method for identifying living microorganisms, it does have drawbacks which make it difficult to use routinely in medical clinical reference laboratories or physicians' offices. In order to illustrate the difficulties, the outline below of the steps in a typical standard procedure will be useful:
1. Cells from the sample to be diagnosed are adsorbed onto the nitrocellulose filter where the hydridization is to be carried out. PA1 2. Cells are lysed on the filter and the DNA is denatured and fixed to the filter. PA1 3. Under renaturing conditions, the filter is exposed to labelled probe DNA. If the sample contains DNA sequences complementary to those of the probe DNA, the probe DNA will combine with the sample DNA and thereby bind to the filter, which is then washed to remove uncombined DNA. PA1 4. The filter is then analyzed for binding of probe DNA by detecting the label on the probe DNA. In most research applications the label is P-32, and it is detected using autoradiography by overlaying the filter with P-32 sensitive film and observing dark spots on the film where the P-32 label has hybridized to the sample DNA.
While this procedure is adequate for research use, it requires considerable skill to carry out and is time consuming. For example, the user must typically carry out a many-step procedure just to adsorb the samples on the filter, to lyse the cells, and to fix the sample DNA on the filter (see Alwine et al, 74 Proc. Natl. Acad. Sci., U.S.A., 5350-56 (1977)). In addition, other steps, including many washings, are required to renature the target DNA in the sample with the labelled probe and to wash away all the unreacted probe.
Fairly expensive specialized equipment and many user steps are involved in detecting the P-32 label by autoradiography. Employing P-32 label limits the use of the test because most physicians and many clinical reference laboratory personnel are not trained in or licensed for the use of this radioactive isotope. In addition, radioactive isotopes can be dangerous to the user and are not easily disposed of.
Besides the several user steps required to spot several samples on a filter, having the user spot the samples can lead to unwanted variability in the test. Amounts of sample spotted and the location of the spot on the filter can be variable. This can lead to difficulty in quantitating final results and in automating the procedures, since most automation procedures would require a precise location of the spots on the planar filter.
Some of the difficulties of the typical standard hybridization procedure can be overcome by using the so-called sandwich hybridization procedures. Typically, such procedures utilize two DNA or RNA probe molecules whose base sequences are complementary to the base sequences of adjacent segments of the target DNA or RNA of the organism to be detected. By using two probes the target DNA becomes divalent, analogous to typical antibody molecules. Thus, many diagnostic kit configurations which have been developed based on the divalency of antibodies are then theoretically adaptable to nucleic acid hybridizations. In such test configurations, the target DNA is "sandwiched" between the two probe molecules. Furthermore, the unique properties of nucleic acids allow for test configurations not possible with antibodies.
In addition to the divalency conferred upon the target DNA in sandwich hybridizations, other advantages result. For example, one of the probes (P.sub.1), can be bound to the planar filters by the manufacturer of the diagnostic kit. Thus, location of the spot on the filter, the area which the spot covers, and the amount of probe bound to the spot can be carefully controlled by the manufacturer to help eliminate variability in the test results and to aid in automating the tests. The precise spotting of P.sub.1 on the filter then directs precisely the target DNA carrying a labelled P.sub.2 probe DNA to the filter spot. Furthermore, the manufacturer has carried out many of the user's steps, making the test easier to use.
Sandwich hybridizations, however, do appear to have at least one major disadvantage. In order for the labelled P.sub.2 probe to bind to the filter, two reactions must take place: the reaction of probe P.sub.1 with target DNA and the reaction of the target DNA with the probe P.sub.2. The necessity of two reactions can slow nucleic acid hybridizations considerably, and any method developed to increase significantly the rate of reaction must apply to both reactions, since the overall rate of reaction is approximately the rate of the slowest of the two reaction steps. This is the well-known "bottleneck" principle of chemical kinetics.
In molecular biology research, nucleic acid hybridizations utilizing sandwiches as described above were first described by Dunn and Hassell, 12 Cell 23 (1977), where a sandwich method was used to detect viral nucleic acid sequences.
Sandwich hybridization tests utilizing radioactive labels have been used to detect viral and bacterial genes are described in Ranki et al., U.S. Pat. No. 4,563,419, and Ranki et al. U.S. Pat. No. 4,486,539, which are hereby incorporated by reference.